Abstract：
ChIP-exo is a powerful tool for achieving enhanced sensitivity and single-base-pair resolution of transcription factor (TF) binding, which utilizes a combination of chromatin immunoprecipitation (ChIP) and lambda exonuclease digestion (exo) followed by high-throughput sequencing. ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode, and single ligation) is an updated and simplified version of the original ChIP-exo method, which has reported an efficient adapter ligation through the DNA circularization step. Building upon an established method, we present a protocol for generating NGS (next-generation sequencing) ready and high-quality ChIP-nexus library for glucocorticoid receptor (GR). This method is specifically optimized for bone marrow-derived macrophage (BMDM) cells. The protocol is initiated by the formation of DNA-protein cross-links in intact cells. This is followed by chromatin shearing, chromatin immunoprecipitation, ligation of sequencing adapters, digestion of adapter-ligated DNA using lambda exonuclease, and purification of single-stranded DNA for circularization and library amplification.
摘要：
ChIP-exo是实现转录因子（TF）结合的增强灵敏度和单碱基对分辨率的强大工具，它利用染色质免疫沉淀（ChIP）和λ外切核酸酶消化（exo）的组合，然后进行高通量测序。ChIP-nexus（通过外切核酸酶进行核苷酸分辨率的染色质免疫沉淀实验，独特的条形码，和单结扎)是原始ChIP-exo方法的更新和简化版本，它已经报道了通过DNA环化步骤的有效衔接子连接。在既定方法的基础上，我们提出了一个方案,用于生成NGS(下一代测序)现成且高质量的糖皮质激素受体(GR)ChIP-nexus文库.该方法特别针对骨髓衍生的巨噬细胞(BMDM)细胞进行了优化。该方案由完整细胞中DNA-蛋白质交联的形成启动。然后是染色质剪切，染色质免疫沉淀，测序衔接子的连接，使用λ外切核酸酶消化衔接子连接的DNA，以及用于环化和文库扩增的单链DNA的纯化。
